Abstract: Objective To investigate the effect of TiO2-NPs, on the osteoblasts and attempt to elucidate the damage response pathways and molecular mechanism to provide scientific basis for developing the new titanium-based implantation. Methods To establish a co-culture system of MC3T3-E1 cells and TiO2-NPs to study the cytotoxic effect of TiO2-NPs on the osteoblasts, the Oxidative stress reaction, apoptosis and possible molecular mechanism. Results The results demonstrated that TiO2-NPs could decrease MC3T3-E1 cells viability, trigger oxidative stress, and activate the p53/bax/bcl-2 -mediated pathway to regulate the apoptosis of osteoblasts. Conclusion TiO2-NPs could cause osteoblasts cytotoxicity in dose-dependent manner and cause oxidative stress reaction and P53 regulated apoptosis. These data demonstrated that TiO2-NPs might have a negative impact on the osteoblasts and provide a theoretical basis to evaluate the biosafety of TiO2-NPs for the design of future materials for implantation.